Man's quest for understanding about life has inspired scientists to explore more deeply into life's biochemical origins. Entire disciplines have developed around obtaining a better understanding of molecules of biological significance including DNA (deoxyribonucleic acid), RNA (ribonucleic acid), proteins, and their respective component molecules. A project to determine the entire human genome was recently initiated by the National Institutes of Health. The eventual goal is to map and sequence the human genome, as well as the genomes of other organisms widely used in research. With such a map, scientists will be better equipped to pinpoint the causes or contributing factors for disease. Once a cause has been determined, the cures are often not far behind.
For this and other reasons researchers are continually looking for ways to manipulate nucleic acids. New methods and devices which can be used to separate, isolate and detect nucleic acids help to drive advancements in the detection and treatment of genetic disorders. Each new development in the ability to manipulate genetic processes has its own characteristics which researchers can exploit to advance in directions which were not before possible.
Scientists have already determined that numerous diseases are linked to defects in the molecules of biological significance, whether at the genetic level (DNA) or at the point of assembly of the final product molecules (i.e., proteins). If the defect occurs in the genetic material itself, one proposed form of therapy involves treatment through the administration of non-defective genetic material. Recently, the first patient was authorized to receive human gene therapy. The patient has Severe Combined Immunodeficiency Disorder (SCID), which has been traced to a defective gene. The therapy involves the infusion of the patient's own white blood cells, after the cells have been genetically engineered to contain a correct version of the defective gene. A recent report indicated that the infused cells were thriving and the patient was doing well. Aside from the obvious benefits, gene therapy provides other treatment advantages over prior methods for treating SCID. Prior treatment methods confined patients to a completely sealed, sterile environment. (Genetic Technology News, Vol. 11, No. 1, p. 13 (January, 1991)). Additionally, the Food and Drug Administration has given the National Cancer Institute permission to treat fifty patients suffering from metastatic melanoma with genetically engineered immune system cells that specifically home in on tumors. (Genetic Technology News, Vol. 11, No. 1, p. 8 (January, (1991)).
As the use of gene therapy increases, so does the demand for properly coded, non-defective genetic material. Current means for DNA synthesis are plagued with some limitations, as well as potential health concerns. One method employs recombinant technology. Essentially, the desired genetic sequence is isolated and inserted into bacterial plasmids via recombinant techniques. As the bacteria reproduce, more copies of the sequence of interest are also made. Bacteria with the proper sequence insertions are then selected through manipulation of antibiotic resistivity. The incorporation of foreign DNA into bacterial plasmids and manipulation of antibiotic resistivity raises some concerns about the development of "super-bugs" which cannot be combatted with currently available treatments and the effect these "bugs" might have if released into the environment. Furthermore, the recombinant DNA process itself is quite laborious and time-consuming for each preparation of a specific DNA sequence.
Another technique disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202 is said to enable the amplification, detection, and cloning of nucleotide sequences. The technique depends upon the detection of specific nucleic acid sequences in a sample, using an oligonucleotide primer specific for the desired sequence, and then subjecting the primer-bound material to conditions favoring synthesis of a complementary strand. The method is dependent upon the presence of the sequence of interest in the sample. If the sequence is not present, or has been somehow modified, amplification of the desired sequence may not proceed.
A third technique disclosed permits the synthesis of oligonucleotides independent of bacteria and plasmids. However, this technique may place limitations upon the length of the nucleotide sequence which may be ultimately synthesized. The technique utilizes a combination of solid-phase chemistry, photolabile protecting groups, and photolithography, and it is claimed that it can be applied to any solid phase synthesis technique in which light can be used to generate a reactive group. The synthesis occurs perpendicular to a glass substrate. Photomasking techniques are used to selectively expose areas of molecules on the substrate containing photolabile reactive groups. The focus appears to be upon synthesizing a large number of molecules of limited chain length, as opposed to molecules of genomic chain length, and the length of the molecules synthesized is evidently determined by the number of synthesis steps performed. The greatest number of synthesis steps disclosed is about 20, which would result in a sequence of about 20 nucleotides. (See Fodor, et al., "Light-directed, spatially addressable parallel chemical synthesis", Science 251:767-773 (1991.)) Since an average-sized gene consists of 1200 base pairs (i.e., 1200 nucleotides on each strand), this technique would require 1200 discreet steps for synthesis of one average-sized gene.
Patients may also suffer adverse consequences when they have inoperative protein molecules, or the complete absence of a particular protein. In such cases, therapy often involves the administration of the operative protein. SCID is an inoperative protein-induced disease and involves a defective enzyme. Protein therapy often precedes gone therapy in time, as it is a prior step in the diagnostic process. First, the cause of a disease may be narrowed down to the failure of a particular protein, or perhaps its absence. Next, the reason for the failure of the protein is determined. Often, this failure can be traced back to a defect in the gene coding for the protein. In the past, genetically engineered microorganisms have been used to produce the operative proteins needed. Examples of such genetically engineered proteins include insulin and Factor VIII. The use of genetically engineered microorganisms presents the same concerns as discussed for the synthesis of DNA using genetically engineered microorganisms--the long-term effects of creating genetically altered "bugs" is simply not known.
Furthermore, the utility of specific, synthesized, DNA sequences does not lie solely in gene therapy. DNA cloning techniques are enjoying widespread application in such diverse fields as identifying crime suspects, identifying fathers in paternity suits, or identifying potential donors for transplants.
Due to the increasing demand for biological molecules, as a result of their extensive application in human therapy and in medical and non-medical analyses, a safer, more efficient method for synthesizing and isolating these molecules is needed.